# EPA/CSPA Technical Meeting on the OECD Method and QACs August 10, 2011 at the EPA ESC

1 AM: 8- 12
• Introductions, scope, agenda – 10 min • Project Leads and Points of Contact • Meeting Minutes • Impact of potential changes to the OECD method on the approval process, the collaborative study, and implementation – 20 min • Highlights of the QAC/OECD Method Data to Date (1 hr)

• Industry data • EPA Data • Problem Definition • Review of the Research Priority List (30 min) • Break – 15 min • Lab demonstrations and discussion (1.5 hr) • Carrier Screening • Hard Water Preparation • Test Microbe Harvest • Neutralizer Confirmation Method • Alternative Procedures (modifications) • Upcoming OECD Test Guidelines Meeting – discuss during lunch PM: 1-5
• Down -select Research Projects – 1.5 hr • List and Prioritize (the top 3 -4) • Test Microbes • Order • SOPs and Modifications • Research Study Protocols • Break – 15 min • Aspects of the Test Designs – 1 hr • Product Treatments • Target Performance Standard • No. of Replications • Controls • Number of Laboratories • Data Analysis • Responsibilities – 15 min • Organizations/Laboratories • Study Matrix by Project • Timeline/reporting – 15 min • Additional Update Meetings – 15 min2 

# DISCLAIMER This information is distributed solely for the purpose of pre-dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency Determination or Policy . The information provided should not be cited or referenced. For Official Use Only/Do not Cite or Distribute

# OECD Quantitative Method Background

3 

## • The OECD Quantitative Method for Evaluating Bactericidal Activity of Microbicides (OECD method) recently underwent an international ring trial. • EPA is interested in adopting the OECD methods for regulatory purposes and is pursuing implementation. • Based on the OECD guidelines, MLB developed and posted SOP MB- 25 (OECD Quantitative Method for Evaluating Bactericidal Activity of Microbicides). • MLB is conducting demonstration studies. • Collaborative study • Implementation Workshop4 

# OECD Quantitative Method Key Features

• Quantitative and “closed” to avoid wash- off of viable microorganisms. • Carriers - brushed stainless steel disks. • Default diluent – 375 ppm hard water. • Contact times that reflect the realities of field applications.

• No more than 5 minutes for vegetative bacteria, mycobacterium, viruses, and fungi . • 20 2oC as default temperature. • Includes a standard soil load. • 5% mixture of high molecular weight protein, low molecular weight protein and mucin • Uses surrogates for all classes of microorganisms.

# OECD Quantitative Method Key Features

1 cm brushed stainless steel disk 10 μL dried inoculum 50 μL disinfectant Vial with inoculated carrier

5 Count colonies and determine log density for control and treated carriers.

# OECD Quantitative Method Demonstration Study



# • Objectives: 1. Gain working experience. 2. Evaluate the method in -house. 3. Generate quantitative efficacy data for EPA- registered products. 4. Assess the technical quality and clarity of MLB SOP MB - 25. 6

# OECD Quantitative Method Demonstration Study

• Evaluated 13 registered liquid antimicrobial products • Represent five common classes of active ingredients • 12 hospital disinfectants,1 sanitizer 

### • Evaluated products against Staphylococcus aureus and Pseudomonas aeruginosa

• OECD parameters (5 min., 375 ppm hard water, 3 -part organic soil load) 

### • 1 replication per product • 3 treated carriers, 4 control carriers • The control target range was 5.5 to 6.5 logs per carrier; assumes a 5 log performance standard. 7

# OECD Quantitative Method Demonstration Study

• To calculate LR values: • Log density (LD) of the control carriers minus LD treated carriers 

### • To provide a LR estimate in cases where the dilutions (for treated carriers) were too numerous to count (e.g., lawns)

• 1,000 was substituted at the least concentrated dilution • The LR was then estimated as a value ≤ LD of control carriers – log 10 

### (1,000) • In treated carrier sets with complete kill, the LR is listed as ≥ average carrier count LD. 8

# OECD Quantitative Method Demonstration Study

• Staphylococcus aureus • Controls: 6.7 0.1 • Carrier counts were slightly above the prescribed upper limit in the OECD method. • In future testing, dilution of the S. aureus culture (e.g., 1:1, 1:5) prior to carrier inoculation will be necessary. 

# • Pseudomonas aeruginosa • Controls: 6.1 0.2 9* *

† * * * * *

† †

† * * * *

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

A B C D E F G H I J K L MLog10Reduction Log Reduction Values for S. aureus using Label Claim and OECD Test Parameters Label Claim OECD

Hydrogen peroxide Phenol Sodium hypochlorite Quaternary ammonium compounds Quat + HCl Citric acid Test Chemicals * = Value listed represents complete kill. † = Value listed represents a value of 1,000 that was substituted at the least concentrated dilution because growth on the filter was a lawn.* * *

† † † † * * * * * *

† † † † † * * * *

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

A B C D E F G H I J K L MLog10Reduction P. aeruginosa Log Reduction using Label Claim and OECD Test Parameters Label Claim OECD

Hydrogen peroxide Phenol Sodium hypochlorite Quaternary ammonium compounds Quat + HCl Citric acid Test Chemicals * = Value listed represents complete kill. † = Value listed represents a value of 1,000 that was substituted at the least concentrated dilution because growth on the filter was a lawn.

# OECD Quantitative Method Conclusions

• Control carrier counts were extremely consistent for both test organisms. • Data (recovered bacteria) for treated carriers were consistent within a product. • No data from treated or control carriers considered to be an outlier. 12

# OECD Quantitative Method Conclusions • Based on this study, method performance was deemed acceptable. • Data indicate a range of LR values, depending on product. • LR values for quaternary ammonium - based products were consistently low (< 5). • Minor changes to the SOP are necessary including the need for preliminary testing. 13

# OECD Quantitative Method Next Steps

• To further verify method performance, MLB recommends a multi - lab evaluation/validation -style study. • OECD method currently being used to test Mycobacterium terrae against tuberculocidal chemicals. • Final decisions on performance standards are pending. 14

# OECD Quantitative Method Collaborative Study Design
• Use MLB SOP MB- 25 • 8- 10 labs with previous training • 6 products, across actives • OECD default parameters • 4 or 5 log performance standard • Two microbes • Test three products per day • Three reps • Kick-off in June 1516 

### Organism **Replicate Test Day**  **Test Chemical**

S. aureus 1 1 D, C, F 2 E, B, A 2 3 B, D, E 4 F, A, C 3 5 B, F, D 6 C, A, E P. aeruginosa 1 7 C, B, D 8 F, E, A 2 9 F, B, D 10 A, E, C 3 11 F, E, C 12 A, D, B 

# OECD Quantitative Method Collaborative Study Design

# OECD Quantitative Method Technical Discussion and Laboratory Worksho

# p

• Proposed for Summer 2011 • Provide updates on the UDM and conduct associated laboratory demonstrations

• Minimum and maximum carrier counts • Performance standards • Further standardization • Phase-out of the UDM 

### • OECD Method Implementation • Laboratory demonstrations • Regulatory status • Science Advisory Panel • Timeline for implementation 17

# OECD Quantitative Method

18 

# Questions/Comments?Treatment Brushed Smooth Total Processed Controls 4 4 20

QAC- 1 3 3 Bleach 3 3 Treatment Brushed Smooth Total Processed

Controls 4 4 20

QAC- 2 3 3 Bleach 3 3 Treatment Brushed Smooth Total Processed

Controls 4 4 20

QAC- 3 3 3 Bleach 3 3 

# Basic design for brushed versus smooth carriersTreatment Medium 1 Dil. TSB Total Processed Controls 4 4 20

QAC- 1 3 3 Bleach 3 3 Treatment Medium 1 Dil. TSB Total Processed

Controls 4 4 20

QAC- 2 3 3 Bleach 3 3 Treatment Medium 1 Dil. TSB Total Processed

Controls 4 4 20

QAC- 3 3 3 Bleach 3 3 

# Basic design for Medium 1 & 2 vs. Diluted TSB (P. aeruginosa)Treatment 1 mL with brushed Total Processed Controls 4 14

QAC- 1 3 QAC- 2 3 QAC- 3 3 Bleach 3 Treatment 1 mL with smooth Total Processed

Controls 4 14

QAC- 1 3 QAC- 2 3 QAC- 3 3 Bleach 3 

# Basic design for Medium 1 vs. Diluted TSBTreatment Default Conditions 10 min/DI/no OSL Total Processed Controls 4 4 20

QAC- 1 3 3 Bleach 3 3

Treatment Default Conditions 10 min/DI/no OSL Total Processed

Controls 4 4 20

QAC- 2 3 3 Bleach 3 3 Treatment Default Conditions 10 min/DI/no OSL Total Processed

Controls 4 4 20

QAC- 3 3 3 Bleach 3 3 

# Basic design for two test parameter sets